Methods have been described for antifungal susceptibility testing (reviewed in McGinnis and Rinaldi, 1991; Warnock, 1989; LaRocco, 1991; Pfaller and Rinaldi, 1993) *see References section herein!. Several reports describe colorimetric methods for antifungal susceptibility testing. One method (Fisher and Armstrong, 1977) determines endpoints after 18 h of incubation by the change in a pH indicator as a result of the production of fungal metabolic acids. Other so-called colorimetric tests (McGinnis and Rinaldi, 1991) either determine cell turbidity at a specific visible light wavelength (Cheung et al., 1975) or measure power-time values with microcalorimetry (Beezer and Chowdhry, 1981).
A recent modification of the NCCLS (National Committees for Clinical Laboratory Standards, 1992) proposed standardized test for antifungal susceptibility testing of yeasts (document M27T) involves determination of end-points by conversion of an oxidation-reduction chromogenic indicator (Pfaller et al., 1994; Pfaller and Barry, 1994). This colorimetric method has provided clearer endpoints for assessment of azole susceptibilities but still requires between 24 to 72 h for a final result. None of the methods involves direct measurement of substrate consumption.
U.S. Pat. No. 4,168,206 to Boyer discloses a method for testing the susceptibility of fungi to antifungal agents and comprises inoculating a non-nutrient agar with a fungus. A number of disks are impregnated with various types and concentrations of antifungal agents in the proper nutrient medium for the type of fungus and antifungal agent tested. The disk is placed upon the non-nutrient medium inoculated with the fungus. After an incubation period the plates are read to determine the minimum inhibitory concentration of the antifungal agent for the particular fungus.
U.S. Pat. No. 4,898,813 to Albarella et al. discloses catalytic test compositions and devices which are capable of generating different color hues at different analyte concentrations. The patent discloses analyte detection wherein the analyte may be glucose. An enzymatic reaction using glucose oxidase is involved. The presence of glucose is detected by a change in color hue.
U.S. Pat. No. 4,254,220 to Meiattini discloses a composition for the kinetic determination of glucose. The composition comprises glucose oxidase and peroxidase enzymes and determines the reaction velocity per concentration unit of glucose.
U.S. Pat. No. 5,091,307 to Escarguel et al. discloses a process characterized by enzymatic reactions which are carried out under anaerobic conditions between a liquid growth medium for mycoplasma containing a dilution medium of the sample of fluid to be analyzed, a first substrate which may be glucose in the presence of a color pH indicator, and a second substrate comprising glucose in the presence of a color indicator. The speed of the enzymatic response is followed while noting the time corresponding to the color change of the indicators. Colony counting is by light microscopy.
U.S. Pat. No. 5,212,066 to Albarella et al. discloses a diagnostic reagent system for the detection of substances which react with peroxidatively active substances resulting in the liberation of hydroperoxides. The reagent comprises a chromogenic oxidation indicator. The patent discloses that the reaction change of glucose with the enzyme glucose oxidase may be monitored.
U.S. Pat. No. 5,312,762 to Guiseppi-Elie discloses the measuring of the presence of an analyte such as glucose. Electrical resistance is measured in the absence and presence of the analyte. The reaction of glucose with glucose oxidase produces hydrogen peroxide which oxidizes a polymer film to make it more conductive. The background of the patent indicates that enzyme assays have been used to monitor therapeutic drugs.
U.S. Pat. No. 4,259,442 to Gayral discloses a method of rapid identification of different species of Streptococcus. A culture specimen is distributed in a certain number of capsules containing supporting disks impregnated with various reactive substrates. Cultures are incubated after which there is deposited on each of the supports a reagent which gives a colored product of reaction with the enzymatic degradation product or products of the substrate. The colors are observed and used to classify the bacterium as one of the Streptococcus groups.
U.S. Pat. No. 3,935,073 to Waters relates to a method and apparatus for detecting biological activity in which the presence of microorganisms is suspected. A growth medium is used containing radioactivity which when fermented produces a radioactive gaseous product. A fermentable carbon source such as glucose is broken down to form CO.sub.2 during the growth of the bacteria. The content of radioactive CO.sub.2 evolved is used to measure bacterial presence and growth in the medium.
U.S. Pat. No. 3,216,907 to Goldman relates to a rapid micromethod for determining the sensitivity of microorganisms to antimicrobial agents. Goldman used glucose detection to determine if bacterial agents in clinical specimens are inhibited by antibacterial drugs. For example, an antibiotic and a pH indicator are added to glucose broth prior to inoculation with the microorganism. The pH indicator will change its color as the pH of the medium drops only when the microorganism is not affected by the antibiotic. The patent indicates that in the presence of minute quantities of glucose, the enzyme glucose oxidase will immediately destroy the glucose to form gluconic acid and hydrogen peroxide. The liberated hydrogen peroxide reacts with a catalyst chromogen or glucose indicator to produce a blue to purple color.
The Goldman assay is not quantitative, thus, MIC determinations would be impossible. He uses infectious material from a patient as the inoculum. The number of infectious agents per sample in this assay are unknown and more than one kind of infectious agent may be present in infectious material. Both of these unknowns preclude quantitation and MIC determination from the Goldman assay.
Coleman et al. (1989) New fluorescence assay for the quantitation of fungi, J. Clin. Microbiol. 27:2003-2007 discloses the exposure of fungi to Fungiqual, a fluorescent stain. The fungi are quantified with a fluorometer which measures the fungiqual fluorescence intensity.
Fisher et al., (1977) Rapid microdilution-colorimetric assay for yeast susceptibility to fluorocytosine, Antimicrob. Agents Chemother. 12:614-617 discloses a yeast assay. The generation of acid by the yeast metabolism of glucose is measured by a change in color.
Hazen et al. (1994) discloses "Potential use of the BacT/Alert automated blood culture system for antifungal susceptibility testing", in J. Clin. Microbiol. 32:848-850. The BacT/Alert system detects growth of fungi or yeast on the basis of chromogenic change in a CO.sub.2 sensitive indicator. The susceptibility of fungi or yeast to antifungal agents is measured with this system.
Hopfer et al. (1977) discloses "Amphotericin B susceptibility testing of yeasts with a Bactec radiometric system" in Antimicrob. Agents Chemother. 11:277-280. In the assay, the metabolism of glucose by yeast is measured by a decrease in a radiolabeled CO.sub.2 production which is measured by a Bactec 225 automated bacterial detection system.
These tests differ from the present invention in several ways. First, no test is presently available that directly measures substrate consumption. Second, only a few tests require less than 24 h to obtain an endpoint and result. The known tests determine endpoints by methods that involve either direct determination of cell mass; cell viability by fluorescence; indirect determination of cell mass by calorimetry, ATP production, or radiometry. Third, rapid tests (&lt;24 h) that have been developed require expensive equipment (Hazen et al., 1994; Beezar and Chowdhry, 1981; Green et al., 1994; Coleman et al., 1989; Anselm and Nilsson, 1984; Hopfer and Groeschel, 1977; Hopfer et al., 1979). Most other published assays utilize reduced fungal growth as the indicator of antifungal activity. Many other methods require either a minimum of two days for results or expensive instrumentation and none measure lethality of the antifungal or antibacterial agent.
The fungal/antimicrobial susceptibility assay of the invention overcomes the deficiencies of prior art fungal susceptibility assays by providing for an assay which measures minimum inhibitory concentration, and can be performed in about 8 hours or less and which involves inexpensive reagents and requires only modest, common clinical laboratory equipment.